Method of increasing the efficacy of antibiotics by compexing with cyclodextrins

ABSTRACT

The present invention provides methods of increasing the biological activity of a bioactive agent by complexing the bioactive agent with a complexing agent. In one preferred embodiment, the bioactive agent is an antibiotic and the complexing agent is a cyclodextrin. However, the invention may be extended to include any drugs as bioactive agents. In certain preferred embodiments, the bioactive agent fits into a hydrophobic core of a complexing agent.

PRIORITY INFORMATION

[0001] This Application is related to the subject matter in U.S. pendingapplication Ser. No. 09/543,964 filed Apr. 7, 2000 and claims benefit ofU.S. Provisional Application No. 60/241,054, filed Oct. 17, 2000. Thecontents of these applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Antibiotics are widely used in medicine for the treatment ofinfections caused by susceptible microbiological organisms. Many ofthese drugs have toxic side effects and/or require increased doses fortreatment of certain infections. Therefore, there exists the need todevelop a system to improve the biological activity of anti-microbials.The applicants have discovered that the biological activity of abioactive agent may be increased by complexation with cyclodextrin. Thecyclodextrin-complexed bioactive agent may be administered to treatinfection caused by a susceptible microorganism.

SUMMARY OF THE INVENTION

[0003] The present invention relates to the discovery that complexationof a bioactive agent to a complexing agent increases the biologicalactivity of the bioactive agent. Thus, the present invention providesmethods of increasing the biological activity of a bioactive agent bycomplexing the bioactive agent to a complexing agent. In certainpreferred embodiments, the bioactive agent is an antibiotic and thecomplexing agent is a cyclodextrin. In particularly preferredembodiments, a hydrophobic antibiotic, such as chlorhexidine,tetracycline, tobramycin, or gentamicin, is complexed with acyclodextrin, such as methyl-β-cyclodextrin orhydroxypropyl-β-cyclodextrin, to increase the ability of the antibioticto inhibit microbial growth. Naturally occurring cyclodextrins as wellas modified naturally occurring cyclodextrins and chemically modifiedcyclodextrins may be used in the inventive method.

[0004] The present invention further provides methods of treating amicrobial infection in an animal by administering to the animal aneffective amount of an antibiotic having increased biological activity.According to the teachings of the invention, the biological activity ofthe antibiotic is increased by complexing it with a cyclodextrin. Thoseskilled in the art will appreciate that the inventive methods may beextended to other diseases or disorders that are treatable with theappropriate bioactive agent, the activity of which has been increasedaccording to the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0005]FIG. 1 is a graph showing the effect of complexation ofchlorhexidine to cyclodextrin on growth of bacteria over time (P.gingivalis (left panel); B. forsythus (right panel)). Abbreviationsinclude chlorhexidine (C); chlorhexidine digluconate (Cg);hydroxypropyl-β-cyclodextrin (H); and methylated-β-cyclodextrin (M).

[0006]FIG. 2 is a graph of the inhibition showing the effect ofcomplexation of chlorhexidine to cyclodextrin on the growth of thebacteria P. gingivalis over time. Abbreviations include chlorhexidine(C); hydroxypropyl-β-cyclodextrin (H); and methylated-β-cyclodextrin(M).

[0007]FIG. 3 is a structural diagram of β-cyclodextrin.

[0008]FIG. 4 is a series of tables illustrating the increased biologicalactivity of a chlorhexidine complexed to cyclodextrin compared touncomplexed antibiotics Page 1: Cx=chlorhexidine digluconate,Tet=tetracycline, Well=quantity of reagent added to well, L=zone ofinhibition. In the boxes on page 1 of FIG. 4, L=length of chipcontaining reagent, W=width of chip containing reagent. Page 2 Lr is theradius of the zone of inhibition or lysis minus the size of the chip; Hris the radius of the zone of inhibition or lysis minus the size of thechip; A is the area of the zone of inhibition or lysis. Page 3 shows thepredicted inhibition (Predicted) using the Gordon et al formula; toppanel is chlorhexidine, bottom panel is actisite (tetracycline).

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

[0009] The present invention provides compositions and methods forincreasing the biological activity of a bioactive agent by linking thebioactive agent to a complexing agent. The invention is based on thediscovery that the attachment of an antibiotic to a complexing agent,specifically a cyclodextrin, increases the biological activity of theantibiotic to a level above that of the uncomplexed antibiotic. Theantibiotic/cyclodextrin complexes are suitable for administration to ananimal for treatment of a microbial infection. Thus, the inventionfurther provides methods of treating a disease or disorder in an animal(e.g., a mammal, preferably a human) by administering an effectiveamount of a bioactive agent complexed to a complexing agent, e.g., acyclodextrin. In one preferred embodiment, the invention providesmethods for treating a microbial infection by administering an effectiveamount of a cyclodextrin-complexed antibiotic.

[0010] The data included herein establish that the biological activityof an antibiotic, such as chlorhexidine, can be enhanced by complexingthe antibiotic with a cyclodextrin. Without limiting the mechanism ofthe invention, we propose that the observed enhancement of biologicalactivity is due to an improved interaction of the complexed antibioticwith the bacterial cell wall, which is composed of poly(sugars).Poly(sugars) are likely to interact with the hydrophilic exterior of thecyclodextrin molecule. In addition, an increase in the solubility of theantibiotic may also contribute to the increase in biological activity.

[0011] The data disclosed herein relate to chlorhexidine as a modelbioactive agent, more particularly to chlorhexidine as a modelanti-microbial agent. As but one example, one may use the methods of theinvention to improve the potency of a narrow spectrum of antibiotics,e.g., hydrophobic or lipophilic antibiotics. Alternatively oradditionally, one may render gram-negative-specific antibiotics activetowards gram-positive pathogens. However, the concepts disclosed hereincan be extended to many drugs. Accordingly, the present invention may beused to improve the biological activity of any bioactive agent.

[0012] The data demonstrate that complexation of chlorhexidine to thecomplexing agent cyclodextrin increases the biological activity of theantibiotic by increasing the ability of the antibiotic to inhibit thegrowth of microorganisms. FIG. 1 is a graph charting the change in sizeof the zone of growth inhibition of the bacterium P. gingivalis overtime. The antibiotic being tested (complexed or non-complexed) isapplied to a specific point near or within an area of bacterial growthon an agar plate. The bioactive agent diffuses from the applicationpoint and causes lysis of already growing bacteria, or inhibition ofgrowth of bacteria within the zone of diffusion. The diameter ofbacterial lysis or growth inhibition is a measure of the potency of thebioactive agent. A relatively large zone of lysis or growth inhibitionindicates that the antibiotic is relatively potent, i.e., it can resultin lysis at very dilute concentrations (on the perimeter of the zone ofdiffusion). A relatively small zone of lysis or growth inhibitionindicates a less biologically active agent.

[0013] Cyclodextrin-complexed chlorhexidine causes larger zones ofbacterial growth inhibition than uncomplexed cyclodextrin on both P.gingivalis and B. forsythus (see Example 1). As described above, withoutlimitation, we propose that complexation of chlorhexidine tocyclodextrin may increase the affinity of the antibiotic to the bacteriavia improved interaction with the bacterial cell wall. FIG. 2 similarlydemonstrates the increased biological activity of cyclodextrin-complexedchlorhexidine.

[0014] The spread sheet compares a known commercial system calledActisite (tetracycline delivery from poly(propylene) fibers) with ourcyclodextrin-complexed chlorhexidine and derivatives.

[0015] Complexing Agents

[0016] In preferred embodiments, the complexing agent has a hydrophobiccore and a hydrophilic exterior. A suitable complexing agent isβ-cyclodextrin, which may be methyl-β-cyclodextrin,hydroxypropyl-β-cyclodextrin, or any derivative thereof. Cyclodextrinsare relatively large cyclic carbohydrates that are conical in shape.FIG. 3 illustrates the structure of β-cyclodextrin showing thehydrophobic core and the hydrophilic exterior. This structure can formsupramolecular inclusion host-guest particles, wherein cyclodextrin isthe host molecule and the bioactive agent is the guest molecule residingwithin the hydrophobic core of cyclodextrin. The host-guest complex maybe generated by physically mixing the cyclodextrin molecule with thebioactive agent.

[0017] Those skilled in the art will appreciate that both natural andchemically modified cyclodextrins are readily available in the art andmay be used in the present invention to increase the biological activityof a bioactive agent (see “Comprehensive Supramolecular Chemistry”Volume 3, edited by József Szejtili and Tetsuo Osa, published byElsevier Science Inc., New York, N.Y.). Naturally occurringcyclodextrins include α-, β-, and γ-cyclodextrins (Pagington, Chemistryin Britain, 23:455 (1987); Parrish, Cyclodextins-A Review, SteringOrganics Ltd. Newcastel-Upon-Tyne. England; Szejtli, CyclodextrinTechnology: Topics in Inclusion Science, Kluwer Academic Publishers(1988)). Modifications of natural cyclodextrins can also easily be madeand include, for example, glucosyl-α-cyclodextrin,maltosyl-α-cyclodextrin, glucosyl-β-cyclodextrin, andmaltosyl-β-cyclodextrin.

[0018] Those skilled in the art will further appreciate that manydifferent chemical moieties may be introduced into the cyclodextrinmolecule and such chemically modified cyclodextrins are readilyavailable (see, e.g., Yoahida et al., Int. Pharm., 46:217 (1988); Mulleret al., J. Pharm. Sci. 75(6): June 1986;; Irie et al., Pharm. Res., No.11, p. 713 (1988)). As but one example, chemically modifiedcyclodextrins may be generated by reaction of the hydroxyl groups liningthe upper and lower ridges of the toroid of cyclodextrin with, forexample, methyl, hydroxyethyl, hydoxylpropyl, carboxymethyl, or acetyl.Each cyclodextrin hydroxyl group differs in its chemical reactivity sothat the reaction process produces an amorphous mixture of thousands ofpositional and potical isomers. The hydroxypropyl-β-cyclodextrin systemis a highly complex mixture of various isometric forms of variouslysubstituted β-cyclodextrin derivatives. This property of amorphousnessis important to certain phsiochemical properties of the chemicallymodified cyclodextrins and has beneficial effects on aqueous solubilityand toxicity of the crystalline parent molecule (Yoshida et al., supra;Muller et al., supra; Irie et al., supra; Muller et al., Pharm Res.10:309 (1985)).

[0019] Those skilled in the art will appreciate that both modifiednatural cyclodextrins and chemically modified derivatives ofcyclodextrin may be used to optimize the bioactivity of a bioactiveagent. Formulations may be further optimized by derivatization of acyclodextrin molecule to optimize the concentration, storage,manufacturing requirements, or route of administration of the bioactiveagent.

[0020] Bioactive Agents

[0021] In certain preferred embodiments, the bioactive agents of theinvention are antibiotics. The term antibiotic is used according toTabers Cyclopedic Medical Dictionary, 15^(th) Ed. to describeantimicrobial substances which have the ability to inhibit the growth ofor to destroy microorganisms. These substances are active in dilutesolutions and may be produced in whole or in part by a microorganism orby a synthetic or semi-synthetic method. Hydrophobic antibiotics areparticularly preferred as they will insert into the hydrophobic core ofa complexing agent, e.g., a cyclodextrin.

[0022] Antibiotics that are useful in the present invention includepenicillin derivatives such as penicillin G, penicillin V, penicillin Gbenzathine, ampicillin, anoxacillin, nafcillin, carbenicilllin,dicloxacillin, bacampicillin, piperacillin, ticaricillin, mezlocillinand the like; cephalosporins such as cefazolin, cefadroxil, cephalexin,cefaclor, cefoxitin, cefonicid, ceftizoxime, cefprozil, ceftazidine,cefixime, cefpodoxime proxitel and the like; aminoglycosides such asamikacin, gentamicin, tobramycin, netilmicin, streptomycin and the like;macrolides such as erythromycin and the like; monobactams such asaztreonam and the like; rifamycin and derivatives such as rifampin,rifamide, rifaximin and the like; chloramphenicol, clindamycin,lincomycin; imipenem; vancomycin; tetracyclines such aschloretetracycline, tetracycline, minocycline, doxycycline and the like;fusidic acid, novobiocin and the like; fosfomycin, fusidate sodium,neomycin, bacitracin, polymyxin, capreomycin, colistimethate, colistinand gramicidin. In addition, cyclodextrin may be complexed with morethan one antibiotic and/or combined with other antibacterial agents.Suitable combinations include:

[0023] rifampin+erythromycin

[0024] erythromycin+sulfonamide such as sulfisoxazole

[0025] penicillin+streptomycin

[0026] rifampin+beta lattamin

[0027] rifamin+fluoroquinolones

[0028] rifampin+vancomycin

[0029] rifampin+tetracyclines

[0030] rifampin+timetoprim

[0031] novobiocin+fluoroquinolones

[0032] trimetoprim+sulfonamides

[0033] rifampin+fusidic acid

[0034] rifampin+isoniazid

[0035] rifampin+fosfomycin

[0036] rifampin+clofazmin+dapsone

[0037] rifampin+aminoside

[0038] vancomycin+fusidic acid.

[0039] Many of the antimicrobial drugs are described in RemingtonsPharmaceutical Sciences, 15^(th) Ed., Chapter 64, which is incorporatedby reference.

[0040] The definition of “bioactive agent” according to the presentinvention may be extended to include many other drugs. Based on thediscovery of the invention, those skilled in the art will appreciatethat complexation of cyclodextrin to other bioactive agents may increasetheir biological activity as it increases the biological activity of theantibiotic chlorhexidine. Suitable bioactive agents include but are notlimited to drugs that are proteins, polypeptides, polynucleotides,nucleoproteins, polysaccharides, glycoproteins, lipoproteins, andsynthetic and biologically engineered analogs thereof.

[0041] Testing multiple bioactive agents for this effect would beroutine to the skilled artisan. One would simply compare the biologicalactivity of the complexed and uncomplexed bioactive agent using abiological assay specific for the particular bioactive agent. Such anassay would be standard if the bioactive agent is, for example, acommercially available pharmaceutical drug for which the biologicalactivity has been well-established. An increase in biological activityof the complexed bioactive agent, compared to the uncomplexed bioactiveagent, indicates that the complexing agent increases the biologicalactivity of the bioactive agent.

[0042] Bioactive agents for use in the present invention include anypharmacologically active substances that produce a local or systemiceffect in animals, preferably mammals, or humans. The term thus meansany substance intended for use in the diagnosis, cure, mitigation,treatment or prevention of disease or in the enhancement of desirablephysical or mental development and conditions in an animal or human.Examples of bioactive agents that might be utilized in the methods ofthe invention include literally any bioactive agent, preferablyhydrophobic bioactive agents. Preferably, though not necessarily, thedrug is one that has already been deemed safe and effective for use bythe appropriate governmental agency or body. For example, drugs forhuman use listed by the FDA under 21 C.F.R. §§330.5, 331 through 361;440-460; drugs for veterinary use listed by the FDA under 21 C.F.R.§§500-582, incorporated herein by reference, are all consideredacceptable for use in the present method.

[0043] Classes of pharmaceutically active compounds that can be used inthe practice of the present invention include, but are not limited to,anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants (e.g., cyclosporine), anti-viral substances, enzymeinhibitors, neurotoxins, opioids, hypnotics, antihistamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, anti-hypertensives, analgesics, anti-pyreticsand anti-inflammatory agents such as NSAIDs, local anesthetics,ophthalmics, prostaglandins, anti-depressants, anti-psychoticsubstances, anti-emetics, imaging agents, specific targeting agents,neurotransmitters, proteins, cell response modifiers, vaccines,ribozymes, anti-sense agents, and RNA.

[0044] A more complete listing of classes of compounds suitable fordelivery into cells according to the present invention may be found inthe Pharmazeutische Wirkstoffe (Von Kleemann et al. (eds) Stuttgart/NewYork, 1987, incorporated herein by reference). Examples of particularpharmaceutically active substances are presented below:

[0045] Anti-AIDS substances are substances used to treat or preventAutoimmune Deficiency Syndrome (AIDS). Examples of such substancesinclude, but are not limited to, CD4, 3′-azido-3′-deoxythymidine (AZT),9-(2-hydroxyethoxymethyl)-guanine (acyclovir), phosphonoformic acid,1-adamantanamine, peptide T, and 2′,3′dideoxycytidine.

[0046] Anti-cancer substances are substances used to treat or preventcancer. Examples of such substances include, but are not limited to,methotrexate, cisplatin, prednisone, hydroxyprogesterone,medroxyprogesterone acetate, megestrol acetate, diethylstilbestrol,testosterone propionate, fluoxymesterone, vinblastine, vincristine,vindesine, daunorubicin, doxorubicin, hydroxyurea, procarbazine,aminoglutethimide, mechlorethamine, cyclophosphamide, melphalan, uracilmustard, chlorambucil, busulfan, carmustine, lomusline, dacarbazine(DTIC: dimethyltriazenomidazolecarboxamide), methotrexate, fluorouracil,5-fluorouracil, cytarabine, cytosine arabinoxide, mercaptopurine,6-mercaptopurine, thioguanine.

[0047] Antibiotics are art recognized and are substances which inhibitthe growth of or kill microorganisms. Antibiotics can be producedsynthetically or by microorganisms. As described above, examples ofantibiotics include, but are not limited to, penicillins,aminoglycosides, macrolides, monobactams, rifamycins, tetracyclines,chloramphenicol, clindamycin, lincomycin, imipenem, fusidic acid,novobiocin, fosfomycin, fusidate sodium, neomycin, polymyxin,capreomycin, colistimethate, colistin, gramicidin, minocycline,doxycycline, vanomycin, bacitracin, kanamycin, gentamycin, erythromicinand cephalosporins.

[0048] Anti-viral agents are substances capable of destroying orsuppressing the replication of viruses. Examples of anti-viral agentsinclude, but are not limited to, α-methyl-P-adamantane methylamine,1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide,9-[2-hydroxy-ethoxy]methylguanine, adamantanamine,5-iodo-2′-deoxyuridine, trifluorothymidine, interferon, and adeninearabinoside.

[0049] Enzyme inhibitors are substances which inhibit an enzymaticreaction. Examples of enzyme inhibitors include, but are not limited to,edrophonium chloride, N-methylphysostigmine, neostigmine bromide,physostigmine sulfate, tacrine HCI, tacrine, 1-hydroxy maleate,iodotubercidin, p-bromotetramisole,10-(alpha-diethylaminopropionyl)-phenothiazine hydrochloride,calmidazolium chloride, hemicholinium-3, 3,5-dinitrocatechol,diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II,3-phenylpropargylamine, N₆-monomethyl-L-arginine acetate, carbidopa,3-hydroxybenzylhydrazine HCl, hydralazine HCl, clorgyline HCl, deprenylHCl,L(−)-, deprenyl HCl,D(+)-, hydroxylamine HCl, iproniazid phosphate,6-MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline HCl, quinacrineHCl, semicarbazide HCl, tranylcypromine HCl,N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride,3-isobutyl-1-methylxanthne, papaverine HCl, indomethacind,2-cyclooctyl-2-hydroxyethylamine hydrochloride,2,3-dichloro-a-methylbenzylamine (DCMB),8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride,p-aminoglutethimide, p-aminoglutethimide tartrate,R(+)-,p-aminoglutethimide tartrate, S(−)-, 3-iodotyrosine,alpha-methyltyrosine, L-, alpha-methyltyrosine, D L-, acetazolamide,dichlorphenamide, 6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

[0050] Neurotoxins are substances which have a toxic effect on thenervous system, e.g. nerve cells. Neurotoxins include, but are notlimited to, adrenergic neurotoxins, cholinergic neurotoxins,dopaminergic neurotoxins, and other neurotoxins. Examples of adrenergicneurotoxins include N-(2-chloroethyl)-N-ethyl-2-bromobenzylaminehydrochloride. Examples of cholinergic neurotoxins includeacetylethylcholine mustard hydrochloride. Examples of dopaminergicneurotoxins include 6-hydroxydopamine HBr,1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydro-pyridine hydrochloride,1-methyl-4-phenyl-2,3-dihydropyridinium perchlorate,N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine HCl,1-methyl-4-phenylpyridinium iodide.

[0051] Opioids are substances having opiate like effects that are notderived from opium. Opioids include opioid agonists and opioidantagonists. Opioid agonists include, but are not limited to, codeinesulfate, fentanyl citrate, hydrocodone bitartrate, loperamide HCl,morphine sulfate, noscapine, norcodeine, normorphine, thebaine. Opioidantagonists include, but are not limited to, nor-binaltorphimine HCl,buprenorphine, chlornaltrexamine 2HCl, funaltrexamione HCl, nalbuphineHCl, nalorphine HCl, naloxone HCl, naloxonazine, naltrexone HCl, andnaltrindole HCl.

[0052] Hypnotics are substances which produce a hypnotic effect.Hypnotics include, but are not limited to, pentobarbital sodium,phenobarbital, secobarbital, thiopental and mixtures, thereof,heterocyclic hypnotics, dioxopiperidines, glutarimides, diethylisovaleramide, a-bromoisovaleryl urea, urethanes and disulfanes.

[0053] Antihistamines are substances which competitively inhibit theeffects of histamines. Examples include, but are not limited to,pyrilamine, chlorpheniramine, tetrahydrazoline, and the like.

[0054] Lubricants are substances that increase the lubricity of theenvironment into which they are delivered. Examples of biologicallyactive lubricants include, but are not limited to, water and saline.

[0055] Tranquilizers are substances which provide a tranquilizingeffect. Examples of tranquilizers include, but are not limited to,chloropromazine, promazine, fluphenzaine, reserpine, deserpidine, andmeprobamate.

[0056] Anti-convulsants are substances which have an effect ofpreventing, reducing, or eliminating convulsions. Examples of suchagents include, but are not limited to, primidone, phenytoin, valproate,Chk and ethosuximide.

[0057] Muscle relaxants and anti-Parkinson agents are agents which relaxmuscles or reduce or eliminate symptoms associated with Parkinson'sdisease. Examples of such agents include, but are not limited to,mephenesin, methocarbomal, cyclobenzaprine hydrochloride,trihexylphenidyl hydrochloride, levodopa/carbidopa, and biperiden.

[0058] Anti-spasmodics and muscle contractants are substances capable ofpreventing or relieving muscle spasms or contractions. Examples of suchagents include, but are not limited to, atropine, scopolamine,oxyphenonium, and papaverine.

[0059] Miotics and anti-cholinergics are compounds which causebronchodilation. Examples include, but are not limited to,echothiophate, pilocarpine, physostigmine salicylate,diisopropylfluorophosphate, epinephrine, neostigmine, carbachol,methacholine, bethanechol, and the like.

[0060] Anti-glaucoma compounds include, but are not limited to,betaxalol, pilocarpine, timolol, timolol salts, and combinations oftimolol, and/or its salts, with pilocarpine.

[0061] Anti-parasitic, -protozoal and -fungals include, but are notlimited to, ivermectin, pyrimethamine, trisulfapyrimidine, clindamycin,amphotericin B, nystatin, flucytosine, natamycin, and miconazole.

[0062] Anti-hypertensives are substances capable of counteracting highblood pressure. Examples of such substances include, but are not limitedto, alpha-methyldopa and the pivaloyloxyethyl ester of alpha-methyldopa.

[0063] Analgesics are substances capable of preventing, reducing, orrelieving pain. Examples of analgesics include, but are not limited to,morphine sulfate, codeine sulfate, meperidine, and nalorphine.

[0064] Anti-pyretics are substances capable of relieving or reducingfever and anti-inflammatory agents are substances capable ofcounteracting or suppressing inflammation. Examples of such agentsinclude, but are not limited to, aspirin (salicylic acid), indomethacin,sodium indomethacin trihydrate, salicylamide, naproxen, colchicine,fenoprofen, sulindac, diflunisal, diclofenac, indoprofen and sodiumsalicylamide.

[0065] Local anesthetics are substances which have an anesthetic effectin a localized region. Examples of such anesthetics include, but are notlimited to, procaine, lidocain, tetracaine and dibucaine.

[0066] Ophthalmics include diagnostic agents such as sodium fluorescein,rose bengal, methacholine, adrenaline, cocaine, and atropine. Ophthalmicsurgical additives include, but are not limited to, alpha-chymotrypsinand hyaluronidase.

[0067] Prostaglandins are art recognized and are a class of naturallyoccurring chemically related, long-chain hydroxy fatty acids that have avariety of biological effects.

[0068] Anti-depressants are substances capable of preventing orrelieving depression. Examples of anti-depressants include, but are notlimited to, imipramine, amitriptyline, nortriptyline, protriptyline,desipramine, amoxapine, doxepin, maprotiline, tranylcypromine,phenelzine, and isocarboxazide.

[0069] Anti-psychotic substances are substances which modify psychoticbehavior. Examples of such agents include, but are not limited to,phenothiazines, butyrophenones and thioxanthenes.

[0070] Anti-emetics are substances which prevent or alleviate nausea orvomiting. An example of such a substance includes, but is not limitedto, dramamine.

[0071] Imaging agents are agents capable of imaging a desired site, e.g.tumor, in vivo. Examples of imaging agents include substances having alabel which is detectable in vivo, e.g. antibodies attached tofluorescent labels. The term antibody includes whole antibodies orfragments thereof.

[0072] Specific targeting agents include agents capable of delivering atherapeutic agent to a desired site, e.g. tumor, and providing atherapeutic effect. Examples of targeting agents include, but are notlimited to, agents which can carry toxins or other agents which providebeneficial effects. The targeting agent can be an antibody linked to atoxin, e.g. ricin A or an antibody linked to a drug.

[0073] Neurotransmitters are substances which are released from a neuronon excitation and travel to either inhibit or excite a target cell.Examples of neurotransmitters include, but are not limited to, dopamine,serotonin, q-aminobutyric acid, norepinephrine, histamine,acetylcholine, and epinephrine.

[0074] Cell response modifiers are chemotactic factors such asplatelet-derived growth factor (PDGF). Other chemotactic factorsinclude, but are not limited to, neutrophil-activating protein, monocytechemoattractant protein, macrophage-inflammatory protein, plateletfactor, platelet basic protein, and melanoma growth stimulatingactivity; epidermal growth factor, transforming growth factor (alpha),fibroblast growth factor, platelet-derived endothelial cell growthfactor, insulin-like growth factor, nerve growth factor, and bonegrowth/cartilage-inducing factor (alpha and beta), or other bonemorphogenetic protein.

[0075] Other cell response modifiers are the interleukins, interleukininhibitors or interleukin receptors, including interleukin 1 throughinterleukin 10; interferons, including alpha, beta and gamma;hematopoietic factors, including erythropoietin, granulocyte colonystimulating factor, macrophage colony stimulating factor andgranulocyte-macrophage colony stimulating factor; tumor necrosisfactors, including alpha and beta; transforming growth factors (beta),including beta-1, beta-2, beta-3, inhibin, and activin; and bonemorphogenetic proteins.

[0076] Uses

[0077] Those of ordinary skill in the art will immediately appreciatethat the present invention can be utilized in a wide variety ofapplications to increase the biological activity of bioactive agents.Bioactive agents having increased biological activity are likely to bemore potent both in vivo and in vitro. For example, a complexedbioactive agent may have an improved effectiveness in a biological assayat the same dose as an uncomplexed bioactive agent. More particularly,bioactive agents, e.g., antibiotics and other drugs, having an increasedbiological activity are useful in vivo for the treatment of disease.Increasing the biological activity of a drug reduces the dose requiredto achieve a desired biological effect. This is advantageous in that theeffective dose of many bioactive agents causes toxic side effects invivo. Side effects may range from mild to severe and may, in some cases,preclude use of the drug. Reducing the dose of the bioactive agent mayresult in reduced in vivo toxicity. Increasing the biological activityof a bioactive agent may also result in improved effectiveness of thecompound in vivo. For example, a maximum dose of a particular antibioticmay have limited effectiveness in vivo for eradicating a particularmicroorganism responsible for an infection. However, the same maximumdose of an antibiotic having increased biological activity due tocomplexation with a cyclodextrin may eliminate the infection entirely.

[0078] Pharmaceutical Compositions

[0079] As will be appreciated by one of ordinary skill in the art,pharmaceutical compositions may be constituted into any form suitablefor the mode of administration selected. Cyclodextrins may beadministered by every route of administration possible. The main causesof toxicity by the cyclodextrins are related to effects on sensitivecell membranes and bulk effects (such as precipitation). Almost allcyclodextrins are safe for topical use because there is no danger of thecyclodextrin getting past the outer skin barrier. Topical uses such aseye drops, suppositories (vaginal and rectal), nasal inhalants, and thelike have the possibility of getting beyond the dermal barrier, but maybe easily tested for irritation and systemic residuals. Bioactive agentsthat are complexed to a cyclodextrin may be easily tested for toxic invivo effects.

[0080] According to the invention, cyclodextrin-complexed bioactiveagents having increased biological activity may be administered in aneffective amount to patients in need of treatment. More particularly,the complexed bioactive agents may be administered in various forms. Forexample, the complexed bioactive agent may be mixed with an excipientand used as an oral administration and/or a non-oral administration.Alternatively, the complexed bioactive agent may be mixed in suitableproportions with supplementary substances such as lubricating agents,emulsifying agents, dispersing agents, and the like.

[0081] For in vivo delivery (i.e., to a patient with a bacterialinfection), it is preferred that the delivery agent be biocompatible andpreferably biodegradable and non-immunogenic. In addition, it isdesirable to deliver a therapeutically effective amount of a compound ina physiologically acceptable carrier. For example, it is known that onemay inject a compound into a patient in a buffered saline solution.Injection into an individual may occur intravenously, intramuscularly,or, for example, directly into a tumor. Alternatively, in vivo deliverymay be accomplished by use of a syrup, an elixir, a liquid, a tablet, apill, a time-release capsule, an aerosol, or a transdermal patch.

[0082] As an oral administration, the complexed bioactive agent may beadministered in the form of a pill, liquid (solutions, syrups, elixirs,and suspensions), powder, tablet, capsule, granules etc. and in suchcases, the excipients used are, apart from water, sugar, starch,dextran, calcium phosphate, calcium carbonate, magnesium oxide,magnesium stearate, aluminium silicate, aluminiium hydroxide, sodiumbicarbonate, glycerin etc. Carriers are intended to include necessaryand inert bindings, suspending agents, lubricants, flavorants,sweeteners, preservatives, dyes, and coatings.

[0083] As a non-oral administration, it may be in the form of aninjection, a drip, an ointment etc. and it may be mixed with a commonsubstance such as distilled water, phsiological saline, vegetable oils,such as olive oil etc., alcohol such as ethanol etc., polyethyleneglycol and so on. Forms useful for parenteral administration includesterile solutions, emulsions, and suspensions.

[0084] Optimal dosages to be administered may be determined by thoseskilled in the art, and will vary with the particular compound in use,the strength of the preparation, the mode of administration, and theadvancement of the disease condition. Additional factors depending onthe particular patient being treated will result in a need to adjustdosages, including patient age, weight, gender, diet, and time ofadministration.

EXAMPLES Example 1 Inhibition of Bacterial Growth Comparison of BacteriaGrowth Inhibition of Drug, Drug Complex, and Complex Components

[0085] Materials

[0086] Trypticase Soy Agar, Becton Dickinson and Company

[0087] Brain Heart Infusion, Becton Dickinson and Company

[0088] Yeast Extract, Becton Dickinson and Company

[0089] MilliQ water

[0090] Hemin (100x), Sigma Chemicals

[0091] Defibrinated Sheep Blood, Binax Laboratories

[0092] Vitamin K, Sigma Chemicals

[0093] N-acetylmuramic acid, Sigma Chemicals

[0094] Teflon well forming device, Massachusetts Institute of Technology

[0095] Electronic Digital Calipers, VWR Scientific

[0096] Hydroxypropyl (H) and methylated beta-cyclodextrin (M), CerestarCompany

[0097] Chlorhexidine (C) and Chlorhexidine Digluconate (Cg), DegussaChemicals

[0098] Poly(dl-lactic-co-glycolic)acid (PLGA), BI Chemicals

[0099]B. forsythus and P. gingivalis, Goodson Laboratory, ForsythInstitute

[0100] Methods

[0101] Increased biological activity of the antibiotic chlorhexidine wastested by placing chips composed of chorhexidine (C) and clorhexidinedigluconate (Cg) complexed with hydroxypropyl-β-cyclodextrin (H) ormethylated β-cyclodextrin adjacent to a tooth in agar seeded with P.gingivalis or B. forsythus. The agar plates were incubated to allowgrowth of the bacterium for 12-15 days and the zone of inhibition ofbacterial growth around each chip was measured. An increase inbiological activity was measured by an increase in the size of the zoneof inhibition around the chip containing the complexed antibioticcompared to the size of the zone around the chip containing theuncomplexed antibiotic.

[0102] More particularly, the method used the following steps:

[0103] Mix: 5 g Trypticase Soy Agar

[0104] 6.5 g Brain Heart Infusion

[0105] 2.5 g Yeast Extract

[0106] 250 mL MilliQ water

[0107] 2.5 mL Hemin (100x).

[0108] Autoclave mixture for 15 minutes at 121 degree Celsius.

[0109] Cool to 50 degrees Celsius for 1 hour.

[0110] Add: 12.5 mL defibrinated sheep blood.

[0111] 2.5 mL Vitamin K (100x).

[0112] 300 uL N-acetylmuramic acid (100x).

[0113] Prepared B. forsythus and P. gingivalis culture solution with anoptical density of 1˜10⁹ cells/mL.

[0114] Added 2 mL of bacteria culture into their respective agarsolutions.

[0115] Pour 20 mL agar solution into 150 mm diameter petri dishes.

[0116] Place well former into petri dishes.

[0117] Allow agar to harden by cooling to room temperature for 1 hour.

[0118] Place petri dishes in anaerobic chamber at 37° C. in anatmosphere consisting of 80% nitrogen, 10% hydrogen, and 10% carbondioxide for 2 days.

[0119] Place 4 mm×5 mm×0.5 mm PLGA chips composed of: Cg, C, C/H, Cg/M,M, and H into preformed wells.

[0120] Masses of each component based on 25 wt % of Cg with respect to a10 mg chip.

[0121] Add MilliQ water into wells to keep chips hydrated.

[0122] Keep petri dishes in anaerobic chamber during entire experiment.

[0123] Measure zones of inhibition daily using electronic calipers fromthe center of the well.

[0124] Experiments done in triplicate.

[0125] Results

[0126] The drug/sugar complex and its individual components were placedon NHK plates of P. gingivalis and B forsythus to compare inhibitionzones. The diameters of the zones of inhibition were measured using anelectronic caliper. PLGA and cyclodextrins were tested foranti-microbial activity individually and demonstrated no inhibition ofthe bacteria species.

[0127] All polymeric chips containing C or its derivatives demonstrateda clear zone of inhibition beginning on the first day. Use of sugarcomplexation resulted in larger zones of bacterial inhibition thanwithout complexation for both periodontal disease causing bacteria, P.gingivalis and B. forsythus. It was observed that polymeric chipscontaining C and Cg showed zones of inhibition that were statisticallysimilar. In both bacterial species studied, polymeric chips composed ofCg/M complex demonstrated the larger zone of inhibition than chipscomposed of C/H.

[0128] As shown in FIG. 1 (left panel) complexation of cyclodextrin tochlorhexidine (hydroxypropyl-β-cyclodextrin or methylatedβ-cylcodextrin) results in approximately a 50% increase in biologicalactivity, which correlates to about a 50% increase in the size of thezone of inhibition of the cyclodextrin-complexed chlorhexidine comparedto the uncomplexed chlorhexidine. Similar result are shown in the rightpanel of FIG. 1, where methylated cyclodextrin-complexed chlorhexidineexhibits about a 30% increase in biological activity compared touncomplexed chlorhexidine (chlorhexidine digluconate). A smallerincrease in the zone of inhibition (from about 40 mm to about 45 mm) wasobserved between hydroxypropyl-β-cyclodextrin-complexed chlorhexidineand uncomplexed chlorhexidine.

BACTERIA INHIBITION STUDY USING NATURAL TEETH

[0129] Materials

[0130] Extracted maxillary 1^(st) to 3^(rd) molars due to large palatalroot surface

[0131] Trypticase Soy Agar, Becton Dickinson and Company

[0132] Brain Heart Infusion, Becton Dickinson and Company

[0133] Yeast Extract, Becton Dickinson and Company

[0134] MilliQ water

[0135] Hemin (100x), Sigma Chemicals

[0136] Defibrinated Sheep Blood, Binax Laboratories

[0137] Vitamin K, Sigma Chemicals

[0138] N-acetylmuramic acid, Sigma Chemicals

[0139] Hydroxypropyl (H) and methylated beta-cyclodextrin (M), CerestarCompany

[0140] Chlorhexidine (C) and Chlorhexidine Digluconate (Cg), DegussaChemicals

[0141] Poly(dl-lactic-co-glycolic)acid (PLGA), BI Chemicals

[0142]P. gingivalis, Goodson Laboratory, Forsyth Institute

[0143] Methods

[0144] Mix: 5 g Trypticase Soy Agar

[0145] 6.5 g Brain Heart Infusion

[0146] 2.5 g Yeast Extract

[0147] 250 mL MilliQ water

[0148] 2.5 mL Hemin (100x).

[0149] Autoclave mixture for 15 minutes at 121 degree Celsius.

[0150] Cool to 50 degrees Celsius for 1 hour.

[0151] Add: 12.5 mL defibrinated sheep blood

[0152] 2.5 mL Vitamin K (100x)

[0153] 300 uL N-acetylmuramic acid (100x).

[0154] Prepared P. gingivalis culture solution with an optical densityof 1˜10⁹ cells/mL.

[0155] Added 2 mL of bacteria culture into their respective agarsolutions.

[0156] Pour 20 ml of agar solution into 150 mm diameter petri dishes.

[0157] Extracted molars were autoclaved.

[0158] Sterilized tooth placed in agar during solidification.

[0159] Allow agar to harden by cooling to room temperature for 1 hour.

[0160] Place petri dishes in anaerobic chamber at 37° C. in anatmosphere consisting of 80% nitrogen, 10% hydrogen, and 10% carbondioxide for 2 days.

[0161] Place 10 mg 4 mm×5 mm×0.5 mm PLGA chips composed of C/H and Cg/Mvertically directly adjacent to the tooth root.

[0162] Keep petri dishes in anaerobic chamber during entire experiment.

[0163] Measure zones of inhibition daily using electronic calipers fromthe center of the well.

[0164] Results

[0165] Polymeric chips containing Cg/M complex and C/H complex wereplaced vertically adjacent to a sterilized tooth in agar seeded with P.gingivalis. Zones of inhibition were larger in the chips containing Cg/Mcomplex than C/H complex that progressively increased or was sustaineduntil day 15. The results are shown in FIG. 2. An excellent zone ofinhibition was observed from day 1 that progressively increased or wassustained until day 15.

COMPARISON STUDY WITH ACTISITE

[0166] Materials

[0167] Mueller-Hinton Broth, Sigma Chemicals

[0168]B. cereus, Goodson Laboratory, Forsyth Institute

[0169] Agar, Becton Dickinson and Company

[0170] Teflon well forming device, produced at Massachusetts Instituteof Technology

[0171] Electronic Digital Caliper, VWR Scientific

[0172] Hydroxypropyl (H) and methylated beta-cyclodextrin (M), CerestarCompany

[0173] Chlorhexidine (C) and Chlorhexidine Digluconate (Cg), DegussaChemicals

[0174] Poly(dl-lactic-co-glycolic)acid (PLGA), BI Chemicals

[0175] Actisite: generous gift from P&G/ALZA Corporation

[0176] Methods

[0177] Prepared Mueller-Hinton Broth at 22 g/L of MilliQ water.

[0178] Add 1.5 g of fresh agar to broth.

[0179] Autoclaved for 15 minutes at 121 degrees Celsius.

[0180] Cool to 50 degrees Celsius for 1 hour.

[0181] Prepared B. cereus culture solution with an optical density of1˜10⁹ cells/mL.

[0182] Add 2 mL of B. cereus culture into agar.

[0183] Pour 20 mL of agar solution into 150 mm diameter petri dishes.

[0184] Place well former into petri dishes.

[0185] Allow agar to harden by cooling to room temperature for 1 hour.

[0186] Weigh Cg/M chips and Actisite Fibers at equivalent masses.

[0187] Place chips, fibers, and standards into preformed wells inhardened agar.

[0188] Cover chips with MilliQ water.

[0189] Incubate in anaerobic chamber at 37° C. in an atmosphereconsisting of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide.

[0190] Measure zones of inhibition using electronic calipers from thecenter of the well.

[0191] Sensitive Assay:

[0192] Gordon, J. M., Walker, C. B., Goodson, J. M., and Socransky, S.S. 1980. Sensitive assay for measuring tetracycline levels in gingivalcrevice fluid. Antimicrobial Agents & Chemotherapy 17: 193-198,incorporated herein by reference.

[0193] Results

[0194] Actisite (Tetracycline) fibers and Cg/M complexed chips werecompared at approximately equivalent total and drug masses. As shown inFIG. 4, it was observed that the zones of inhibition for Cg/M complexedchips were more defined than zones of inhibition for the Actisitefibers. Using the Sensitive Assay, calculations predicted that chipsmade of Cg/M complex were more effective at reaching a higher level ofbacterial inhibition.

[0195] Other embodiments of the invention will be apparent to thoseskilled in the art from a consideration of the specification or practiceof the invention disclosed herein. It is intended that the specificationand examples be considered as exemplary only, with the true scope andspirit of the invention being indicated by the following claims.

What is claimed is:
 1. A method of increasing the biological activity ofa bioactive agent comprising the steps of complexing the bioactive agentto a complexing agent, wherein the biological activity of a bioactiveagent/complexing agent complex is higher than the activity of anuncomplexed bioactive agent.
 2. The method of claim 1, wherein thebioactive agent is an antibiotic.
 3. The methods of claim 2, wherein theantibiotic is a hydrophobic antibiotic.
 4. The method of claim 2,wherein the antibiotic is selected from the group consisting oftetracycline, tobramycin, and gentamicin.
 5. The methods of claim 2,wherein the antibiotic is chlorhexidine.
 6. The methods of claim 2,wherein the antibiotic is chlorhexidine digluconate.
 7. The method ofclaim 1, wherein the complexing agent is cyclodextrin.
 8. The method ofclaim 7, wherein the cyclodextrin is a naturally occurring cyclodextrin.9. The method of claim 7, wherein the cyclodextrin is a chemicallymodified cyclodextrin.
 10. A method of increasing the biologicalactivity of an antibiotic against a microbial agent comprisingcomplexing the antibiotic to a cyclodextrin, wherein the biologicalactivity of a cyclodextrin/antibiotic complex is higher than theactivity of an uncomplexed antibiotic.
 11. The method of claim 10wherein the antibiotic is hydrophobic.
 12. The method of claim 10,wherein the antibiotic is selected from the group consisting oftetracycline, tobramycin, and gentamicin.
 13. The method of claim 10,wherein the antibiotic is chlorhexidine.
 14. The method of claim 10,wherein the antibiotic is chlorhexidine digluconate.
 15. The method ofclaim 10, wherein the cyclodextrin is a naturally occurringcyclodextrin.
 16. The method of claim 10, wherein the cyclodextrin is achemically modified cyclodextrin.
 17. A method of treating a microbialinfection in an animal comprising administering to the animal aneffective amount of an antibiotic that is complexed to a cyclodextrin.18. The method of claim 17, wherein the antibiotic is hydrophobic. 19.The method of claim 17, wherein the antibiotic is selected from thegroup consisting of tetracycline, tobramycin, and gentamicin.
 20. Themethod of claim 17, wherein the antibiotic is chlorhexidine.
 21. Themethod of claim 17, wherein the antibiotic is chlorhexidine digluconate.22. The method of claim 17, wherein the cyclodextrin is a naturallyoccurring cyclodextrin.
 23. The method of claim 17, wherein thecyclodextrin is a chemically modified cyclodextrin.